A 3D cell culture is an artificially created environment in which biological cells are permitted to grow or interact with their surroundings in all three dimensions. Unlike 2D environments (e.g. a Petri dish), a 3D cell culture allows cells in vitro to grow in all directions, similar to how they would in vivo. These three-dimensional cultures are usually grown in bioreactors, small capsules in which the cells can grow into spheroids, or 3D cell colonies. Approximately 300 spheroids are usually cultured per bioreactor.
Background
3D cell cultures have been used in research for several decades. One of the first recorded approaches for their development was at the beginning of the 20th century, with the efforts of Alexis Carrel to develop methods for prolonged in vitro tissue cultures. Early studies in the 80's, led by Mina Bissell from the Lawrence Berkeley National Laboratory, highlighted the importance of 3D techniques for creating accurate in vitro culturing models. This work focused on the importance of the extracellular matrix and the ability of cultures in artificial 3D matrices to produce physiologically relevant multicellular structures, such as acinar structures in healthy and cancerous breast tissue models. These techniques have been applied to in vitro disease models used to evaluate cellular responses to pharmaceutical compounds.MERIT Award Recipient: Mina J. Bissell, Ph.D. (n.d.). Retrieved 16 June 2016, from http://www.cancer.gov/research/nci-role/spotlight/merit/Bissell
Eric Simon, in a 1988 NIH SBIR grant report, showed that electrospinning could be used to produce nano- and submicron-scale polystyrene and polycarbonate fibrous mats (now known as scaffolds) specifically intended for use as in vitro cell substrates. This early use of electrospun fibrous lattices for cell culture and tissue engineering showed that various cell types including Human Foreskin Fibroblasts (HFF), transformed Human Carcinoma (HEp-2), and Mink Lung Epithelium (MLE) would adhere to and proliferate upon the fibers. It was noted that as opposed to the flattened morphology typically seen in 2D culture, cells grown on the electrospun fibers exhibited a more histotypic rounded 3-dimensional morphology generally observed in vivo.
3D cell culture, by emulating essential aspects of the in vivo environment, including interactions between cells and the extracellular matrix, allows for the faithful recreation of structural architecture and specialized functions in normal tissues or tumors in a laboratory setting. This approach authentically models the conditions and processes of living tissues, producing responses akin to those observed in vivo. Since its inception in the 1970s, 3D cell culture has provided significant insights into the mechanisms regulating tissue homeostasis and cancer. Moreover, it has expedited translational research in the realms of cancer biology and tissue engineering. To date a major limiting factor in the size of constructs has been unprogrammed cell death called necrosis.
Properties In living tissue, cells exist in 3D microenvironments with intricate cell-cell and cell-matrix interactions and complex transport dynamics for nutrients and cells. Standard 2D, or monolayer, cell cultures are inadequate representations of this environment, which often makes them unreliable predictors of in vivo drug efficacy and toxicity. 3D spheroids more closely resemble in vivo tissue in terms of cellular communication and the development of extracellular matrices. These matrices help the cells to be able to move within their spheroid similar to the way cells would move in living tissue. The spheroids are thus improved models for cell migration, differentiation, survival, and growth. Furthermore, 3D cell cultures provide more accurate depiction of cell polarization, since in 2D, the cells can only be partially polarized. Moreover, cells grown in 3D exhibit different gene expression than those grown in 2D.
The third dimension of cell growth provides more contact space for mechanical inputs and for cell adhesion, which is necessary for integrin ligation, cell contraction and even intracellular signalling. Normal solute diffusion and binding to effector proteins (like growth factors and enzymes) is also reliant on the 3D cellular matrix, so it is critical for the establishment of tissue scale solute concentration gradients
For the purposes of drug toxicology screening, it is much more useful to test gene expression of in vitro cells grown in 3D than 2D, since the gene expression of the 3D spheroids will more closely resemble gene expression in vivo. Lastly, 3D cell cultures have greater stability and longer lifespans than cell cultures in 2D. This means that they are more suitable for long-term studies and for demonstrating long-term effects of the drug. 3D environments also allow the cells to grow undisturbed. In 2D, the cells must undergo regular trypsinization to provide them with sufficient nutrients for normal cell growth. 3D spheroids have been cultured in a lab setting for up to 302 days while still maintaining healthy, non-cancerous growth.
In the interdisciplinary research of biology and aerospace, the 3D printed-scaffolds are also being used for protecting cells from the effect of gravity during the launching.
Classification of 3D culture methods There are a large number of commercially available culturing tools that claim to provide the advantages of 3D cell culture. In general, the platforms can be classified in two types of 3D culturing methods: scaffold techniques and scaffold-free techniques.
Scaffold techniques Scaffold techniques include the use of solid scaffolds, hydrogels and other materials. In a recent study potentiality of human CD34+ stem cells explored by generating in vitro agarose gel 3D model to understand the bone ossification process. Scaffolds can be used to generate microtissue 3D model by culturing fibroblasts outside of tumour cells, mimicking the tumor stroma interaction.
The effectiveness of scaffolds in various applications, particularly in tissue engineering, is significantly impacted by factors such as pore distribution, exposed surface area, and porosity. The quantity and arrangement of these elements influence both the depth and rate at which cells penetrate the scaffold volume, the structure of the resulting extracellular matrix, and ultimately, the success of the regenerative process. Scaffolds can be produced with diverse architectures depending on the manufacturing method, leading to either random or precisely designed pore distribution. Recently, advanced computer-controlled rapid prototyping techniques have been employed to create scaffolds with well-organized geometries.
Hydrogels As the natural extracellular matrix (ECM) is important in the survival, proliferation, differentiation and migration of the cells, different hydrogel matrices mimicking natural ECM structure are considered as potential approaches towards in vivo –like cell culturing. Hydrogels are composed of interconnected pores with high water retention, which enables efficient transport of e.g. nutrients and gases. Several different types of hydrogels from natural and synthetic materials are available for 3D cell culture, including e.g. animal ECM extract hydrogels, protein hydrogels, peptide hydrogels, polymer hydrogels, and wood-based nanocellulose hydrogel.
The approach to crafting the optimal ECM replica relies on the specific characteristics of the culture in question and typically involves employing diverse and independent chemical processes. For example, the utilization of photolabile chemistries can lead to the erosion of specific regions within a gel, and subsequently exposing these areas allows for the application of adhesive ligands, promoting cell adhesion and migration. The development of more intricate frameworks is anticipated, comprising interwoven networks of chemistries under the control of both cells and users. In essence, there is no singular network capable of faithfully emulating the intricate ECM of every tissue type. However, a thoughtful integration of bioinspired cues into synthetic gels holds the potential to yield resilient and versatile scaffolds applicable across various cell culture systems.
Scaffold-free techniques Scaffold free techniques employ another approach independent from the use scaffold. Scaffold-free methods include e.g. the use of low adhesion plates, hanging drop plates, micropatterned surfaces, and rotating bioreactors, magnetic levitation, and magnetic 3D bioprinting.
Spheroids
Spheroids are a type of three-dimensional cell modeling that better simulate a live cell's environmental conditions compared to a two-dimensional cell model, specifically with the reactions between cells and the reactions between cells and the matrix. Due to the strong interactions between the cells and the extracellular matrix in the spheroid, the permeability is limited, generating physiologically relevant gradients of e.g. nutrients and oxygen through the spheroid. Spheroids are useful in the study of changing physiological characteristics of cells, the difference in the structure of healthy cells and tumor cells, and the changes cells undergo when forming a tumor. Spheroids co-cultured with tumor and healthy cells were used to simulate how cancerous cells interact with normal cells. Spheroids can also be co-cultured with fibroblasts to mimic tumor-stroma interaction. Spheroids can be grown with a few different methods. One common method is to use low cell adhesion plates, typically a 96 well plate, to mass-produce spheroid cultures, where the aggregates form in the rounded bottom of the cell plates. Spheroids can also be cultured using the hanging drop method involving forming cell aggregates in drops that hang from the surface of a cell plate. Other methods under investigation include the use of rotating wall vessel bioreactors, which spins and cultures the cells when they are constantly in free fall and forms aggregates in layers Recently, some protocols have been standardized to produce uniform and reliable spheroids. Researchers had also explored standardized, economical and reproducible methods for 3D cell culture. To improve reproducibility and transparency in spheroid experiments, an international consortium developed MISpheroID (Minimal Information in Spheroid Identity).
Clusteroids clusteroids are a type of three-dimensional cell modeling similar to spheroids but are distinguished by their creation method; grown as clusters of cells in an aqueous two-phase system of water-in-water Pickering emulsion using interfacial tension and osmotic shrinkage to pack the cells into dense clusters which are then cultured in a hydrogel into tissues or organoids.
In the absence of blood vessels, oxygen permeability is impaired during necrotic nucleus formation and this prevents the ex vivo use of 3D cell culture. There is an emulsion template that can overcome this problem. This approach allowed researchers to adjust the cell composition to attain the ideal conditions for promoting the synthesis of diverse angiogenic protein markers within the co-cultured clusteroids. HUVEC cells exhibit a reaction to the presence of Hep-G2 cells and their derivatives by generating endothelial cell sprouts in Matrigel, all without the external introduction of vascular endothelial growth factor (VEGF) or other agents that induce angiogenesis. The replication of this cultivation technique is straightforward for generating various cell co-culture spheroids. The w/w Pickering emulsion template greatly aids in constructing 3D co-culture models, offering significant potential for applications in drug testing and tissue engineering.
Bioreactors